Research highlights

Apoptosis: A lipid trigger of MOMP

Standfirst

One of the crucial events of the mitochondrial pathway of apoptosis is mitochondrial outer membrane permeabilization (MOMP), which is carried out by the two B cell lymphoma 2 (BCL-2) family proteins BCL-2 antagonist/killer (BAK) and BAX. These proteins undergo extensive conformational changes to become activated, and Green and colleagues now reveal that this requires sphingolipids originating from heterotypic membranes.

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Because mitochondria form stable contacts with other organelles, such as the endoplasmic reticulum, the authors asked whether heterotypic membranes contain components that may promote MOMP. Indeed, although a highly purified mitochondrial membrane fraction showed low levels of cytochrome c release following BAK activation (triggered by treatment with caspase 8-cleaved BH3-interacting domain death agonist (C8-BID)), release could be restored by the addition of a preparation containing heterotypic membranes.

So what component in the heterotypic membrane fraction activates BAK? The neutral sphingomyelin phosphodiesterases SMPD3 and SMPD4, which were found to be of non-mitochondrial origin, were identified as potential candidates. Inhibition of SMPD activity blocked cytochrome c release, and addition of SMPD3 to membrane fractions enhanced cytochrome c release mediated by C8-BID treatment. Consistently, using a different approach to investigate BAX activation specifically, the authors observed that SMPD3 cooperated with C8-BID to induce MOMP.

SMPDs are part of the sphingomyelin metabolism pathway and hydrolyse sphingomyelin to ceramide, which is broken down to sphingosine, sphingosine 1-phosphate (S1P) and then ortho-phosphoethanolamine and hexadecenal (hex). By testing a panel of inhibitors that target different stages of the pathway, or by reconstituting the pathway with different enzymes and lipids, the authors conclude that S1P is required for C8-BID-induced BAK activation. By contrast, BAX activation requires hex; the two bind directly, and this interaction promotes BAX activation and oligomerization, which in turn leads to MOMP and cytochrome c release.

Finally, the authors examined the requirement of sphingolipids for BAK and BAX activation in a cellular context. Consistent with the above results, expression of SMPD3 enhanced cell sensitivity to tumour necrosis factor (TNF)-induced apoptosis. Moreover, silencing of sphingosine kinase 2 (SPHK2) but not of SPHK1 — both of which break down sphingosine to S1P — made cells more resistant to TNF-induced apoptosis. Importantly, SPHK2 could be detected in mitochondria, which indicates that a sphingosine precursor is exported from the heterotypic membrane and localizes to mitochondria, where it is further metabolized.

The sphingomyelin metabolism pathway had previously been suggested to have a role in apoptosis, and this work reveals how it intersects with known apoptotic mediators. The authors propose that ceramide derived from sphingomyelin in the heterotypic membrane is transferred to mitochondria, where it is metabolized by enzymes such as SPHK2, giving rise to S1P and hex. These then promote BAK and BAX activation, respectively, leading to MOMP and cytochrome c release.